Three‐dimensional morphological revealing of human placental villi with common obstetric complications via optical coherence tomography

Abstract Placental villi play a vital role in human fetal development, acting as the bridge of material exchange between the maternal and fetal. The abnormal morphology of placental villi is closely related to placental circulation disorder and pregnancy complications. Revealing placental villi three‐dimensional (3D) morphology of common obstetric complications and healthy pregnancies provides a new perspective for studying the role of the placenta and its villi in the development of pregnancy diseases. In this study, we established a noninvasive, high‐resolution 3D imaging platform via optical coherence tomography to reveal placental villi 3D morphological information of diseased and normal placentae. For the first time, 3D morphologies of placental villous tree structures in common obstetric complications were quantitatively revealed and corresponding 3D information could visualize the morphological characteristics of the placental villous tree from a more intuitive perspective, providing helpful information to the study of fetal development, feto‐maternal material exchange, and gestational complications treatment.


| INTRODUCTION
Placenta is a feto-maternal organ that provides oxygen and nutrients to the fetus and removes waste products from the fetus' blood. 1,2 The key part of the placenta is the villous tree which sprouts from the chorionic plate into the intervillous space. 3 The branches of the stems continue branching, leading to a large number of stem villi generations and further branches, finally ending as freely floating villi in the intervillous space. Stem villi 4 have the largest diameter, which serves as the mechanical support for the villi tree and plays a small role in the exchange of fetal material. From the intermediate villi branching from the stem villi sprout terminal villi, which are the most important components of the villous tree. 5 Villous trees, acting as part of the border between maternal and fetal blood during pregnancy, increase the area of the chorionic membrane across where oxygen, carbon dioxide, and other substances can diffuse between the maternal and fetal blood. 2 Many pregnancy complications are related to the maldevelopment of the placental villous tree. 6 Material exchanging between maternal and fetus in the placenta cannot provide the fetus with the nutrients needed for normal growth, which is considered to be a possible cause of fetal growth restriction (FGR). 7 Gestational hypertension (GH), one category of hypertensive disorders of pregnancy (HDP) that is one of the leading causes of morbidity and mortality, can be dangerous for both the mother and fetus. Although the cause of GH is remaining unclear now, it has been reported that the placenta villi with GH have abnormalities in histological features. 8 Gestational diabetes mellitus (GDM) is a type of diabetes that develops during pregnancy and causes many structural and functional changes in the placenta villi. 9 Therefore, revealing the three-dimensional (3D) morphology of multidiseased placental villi is of great significance for understanding the interaction and influence of placental villi with gestational complications, as well as for studying the fetal development, feto-maternal material exchange, and gestational complications treatment.
Up to now, three-dimensional (3D) visualizing human placental villous tree structures are limited. Most of our understanding of placental villi is based on histologic analysis 10 of stained thin sections from delivered placenta specimens. However, histological section as a two-dimensional (2D) method, cannot present the 3D morphology of placental villi, and also time-costing. Scanning electron microscopy (SEM) 11 is used to view placenta microstructure subjects to artifacts and has the problems of narrow field of view and limited depth of focus. Confocal laser scanning microscopy (CLSM) 12 which needs to perfuse and stain the sections, like the histological section, is also faced the same limitations. Synchrotron X-ray imaging 13 is used to generate high-resolution massively multiscale datasets of the human placenta. However, it requires complex fixation, perfusion, staining, and embedding operation before imaging. Micro-CT 14 can provide volume imaging with micrometer scale resolution but magnification is at the cost of a limited field of view. The lack of 3D morphological revealing of placenta villi limits our ability to view the villous tree as a whole and characterize many morphological features of the villous tree, such as branches and villi morphology.
Here we proposed a noninvasive, high-resolution optical coherence tomography (OCT) 15 imaging platform to reveal 3D morphological information on the villous tree structure of healthy human placentae and human placentae with common obstetric complications. As an emerging technology for biomedical imaging technology, OCT employing near-infrared light low-coherence interferometry, can achieve non-destructive, micron-level resolution 3D imaging of bio-tissue, 16,17 and has been widely used for biomedical imaging and clinical diagnosis, such as imaging human retinal 3D microstructures in vivo. In this study, we employed a noninvasive 3D imaging platform via OCT to reveal the 3D morphology of multi-diseased placental villi and extracted highresolution structural and morphological information from threecategory gestational complications and normal placentae for the first time. In particular, we quantitatively extracted the morphological characteristics of the placental villous tree structure including branches and villi morphology for each diseased and normal placenta which are significant to study the effect of pregnancy complications on the morphology of the placental villi. Moreover, we also first revealed the villous tree morphology of the two-gestational-comorbidities placenta, providing a new perspective for studying the relationship and mutual influence of gestational com- (4) Regular birth examination during pregnancy; and (5) definite diagnosis of the corresponding disease in the obstetric outpatient clinic.
We collected placentae from six cases of the healthy pregnancy, six cases of the pregnancy with HDP, eight cases of the pregnancy with GDM, and six cases of the pregnancy with FGR including two cases of the pregnancy complicated with FGR and GDM.
The placental tissues were randomly obtained from the maternal surface of the placenta after the placenta delivery in 1 h, as Figure 1 shows. The placental tissues in each case were immersed in 0.9% physiological saline to maintain morphology and immediately transported to the laboratory with ice bags using a biological sample delivery box. Dissected villus tree indicated by the blue dashed box in Figure 1a isolated from delivered placental tissue indicated by the green dashed box in Figure 1a after fetal blood was cleared from the tissue with physiological saline. At least four placental tissues were obtained from each placenta, and more than 20 dissected villus tree regions were isolated from each placenta tissue for 3D imaging. More than 80 villus tree regions were obtained from each placenta sample to ensure the results of 3D imaging of placental villous trees are more general.
To compare the imaging performance of OCT and histological sections, we prepared a healthy placenta sample to implement both OCT and histological sections, as Figure 1b shows. We collected and acquired the placenta sample following the same criteria and transported it to the laboratory. First, we performed OCT imaging of the placenta villi. Placental villous tissue was removed from the 0.9% normal saline, dried on the absorbent paper towel, and then embedded in an optimal cutting temperature medium to acquire OCT images. After OCT imaging, optical cutting temperature embedded placental villous was quickly placed on a freezing microtome for snap freezing. We used Leica Biosystems cryostat to prepare sections and optimal cutting temperature embedded villous were serially frozen-sectioned at 10 μm thick along the longitudinal direction. The sections were first stained with hematoxylin for 10 s and washed with dripping water, then stained with eosin for 150 s and washed again with dripping water for 1-2 s. Different concentration gradients of alcohol were used for dehydration. Next, we used xylene for transparent treatment. Finally, the sheets were blocked with neutral gum. The stained sections were observed under a microscope, and two-dimensional images of a suitable region were acquired.

| 3D imaging platform
Here a spectral-domain OCT system was used as the 3D imaging platform for the placenta villi's three-dimensional morphological revealing, as

| Data processing
OCT images of the villous tree of placenta with three different gestational complications and healthy pregnancy were acquired immediately after delivery and transported from the hospital to the lab room.
OCT scanning range was adjusted to acquire a broad view of villous trees. The actual scanning range was~4:08 Â 4:08 mm 2 , and the corresponding transverse pixel resolutions was~5.1 μm, respectively.
Obtained OCT datasets of villous trees were first processed to generate OCT structural images with rescaling operation in the axial direction to achieve the same pixel resolution as that in the transverse direction. Each 3D morphology image of placenta villi was acquired via the OCT imaging platform and further processed via ImageJ 18 (Version 1.53c, US National Institutes of Health). Here the rescale operation of OCT images was implemented by using the ImageJ scale operation. Here we used a 0.3333 index to rescale the depth axis to achieve the same resolution with the transverse plane to acquire an accurate 3D effect of placenta villi. We used the 'scale' method of ImageJ and adjust the Y scale to 0.3333.  To quantify the number of villi branches, we used 3D OCT images of corresponding villi and villi branches that could easily be observed in 3D images, as Figure 3c shows. For each villus, we counted the branches of it three times and take the average to get the result. We also performed the rotation to count the branch number accurately.
To quantify the length of placental villi, we calculated three crosssectional images in the XY, XZ, and YZ planes, selecting all frames where the selected placental villi appeared, as Figure 3d shows. The length of terminal villi L is approximately calculated as: where F XY , F XZ , F YZ was frame number where the selected placental villi were present in XY, XZ, YZ cross-sectional images, respectively, δ was the rescaled resolution of OCT images.

| RESULTS
Here

| OCT versus histological sections
Here we implemented histology sections on a placenta sample of healthy pregnancy to compare the imaging performance between histological sections and OCT, as

| Healthy pregnancy
Histology method indicates that placental villi from full-term women without comorbidities are densely branched, with tightly packed adjacent villous shafts, and numerous villi. 4 Figure 5a-d and Video S1 reveal the morphology of a placenta of healthy pregnancy. A top view of a 3D OCT image of the normal placental villous tree revealed villi morphology of healthy pregnancy placenta sample, as Figure 5a shows.

| Gestational hypertension
Gestational hypertension (GH) is one of the most common categories of HDP. HDP is a complex diseases category that can be differentiated into many disorders for each will cause different gestational outcomes. 19

| Fetal growth restriction
Fetal growth restriction (FGR) 23 is defined as the failure of a fetus to achieve its genetic growth potential. FGR is an important cause of premature delivery and stillbirth, which is also associated with neonatal morbidity and mortality, impaired health in childhood, 24 and increased rate of coronary heart disease and related disorders, stroke, hypertension, and type 2 diabetes in later life. 25 Placental insufficiency is a major cause of fetal growth restriction, and one of the reasons is abnormalities of placental villous architecture. 26,27 Traditional pathological studies of FGR have suggested that both the weight and its diameter of the placenta are significantly smaller than in healthy pregnancy, with focal necrosis or fibrosis of the villi, and reduced number and diameter of intervillous capillarization, suggesting that primary villous dysplasia may be a potential contributor to fetal growth restriction.
Here we employed our 3D imaging platform to reveal the villi morphology of placentae with FGR.

| Multiple comorbidities of FGR and GDM
During pregnancy, two or more complications occurred simultaneously make the pregnancy more complex to diagnose and treat.
Therefore, it is of practical significance to study the placenta morphology that suffers from multiple pregnancy complications at the same time. Here 3D morphology of a placenta with FGR and GDM was revealed via the OCT imaging platform. shows. Healthy placenta terminal villi have a larger diameter than complicated placental terminal villi except for placenta with GDM.
Placenta samples with GDM have longer terminal villi than healthy and other complicated placenta samples we studied, as Figure 10d shows. These measured data agree with placental villi morphological characters extracted from 3D OCT images and cross-section images before.
The results of placental villi morphological parameters quantification are shown in Table 1. Compared with healthy placenta, mean IVD of complicated placenta show statistically significant

| DISCUSSION AND CONCLUSION
In this study, we established a 3D imaging system via OCT to acquire The current quantitative assessment of placenta villi is conducted manually and quantitative speed and accuracy would be affected by human factors. Quantitative analysis's speed and accuracy can be further improved by combining with artificial intelligence methods. In the future, the method based on artificial intelligence will greatly improve quantitative analyses efficiency and achieve higher precision, which we are focusing on now. At present, the total amount of placental samples in our study is relatively insufficient, and further research will obtain more reliable and accurate results. Here the pixel-resolution rescale operations were performed on the 3D image volumes, and a specific index mentioned in Section 2.3 was chosen to numerically rescale the axial pixel resolution to achieve that the acquired 3D image volumes have the same pixel resolution along different axis directions, so it can be more convenient to observe and measure the 3D morphology of human placental villi. Meanwhile, here we assumed the refractive index of the sample over a wide range of wavelengths as~1.38, following the References 30,31 to estimate the OCT axial optical resolution in the sample, which was~1.7 μm.
In summary, we have revealed and extracted 3D morphological characteristics of multi-diseased and healthy human placenta via our customized OCT imaging system and demonstrated possible influence mechanism of placental villous tree morphology with different gestational complications on feto-maternal material exchange. In addition to villous tree structure visualization, our study provides complementary 3D information to better characterize villous tree structure morphology as a whole, yielding an intuitive and noninvasive morphological revealing of human placenta villi via OCT.
Moreover, our customized 3D imaging system via OCT could be reliably utilized to reveal inner villi structures such as capillary and membrane in villi by using a higher magnification microscope, suggesting a promising study prospect of placental villous capillary morphology.
Overall, OCT not only reinforced the depth-resolved distinct morphologies noted by histological sections but also uncovered key 3D